GSM8083549: GW39_2_Arc; Homo sapiens; RNA-Seq
Frozen sections were transferred from tubes in dry ice to ice cold lysis buffer (0.01M Tris-HCl, 0.14M NaCl, 1mM CaCl2, 0.02M MgCl2, 0.03% Tween-20, 0.01% BSA, 10% Nuclei Ez Lysis Buffer (Sigma), 0.2U/μL Protector RNAse inhibitor (Sigma) in DEPC-treated water) and the tissue was dissociated using a glass dounce homogenizer (Thomas Scientific, Cat # 3431D76). Tissue homogenates were transferred to a separate 30 mL thick wall polycarbonate ultracentrifuge tube (Beckman Coulter, Cat # 355631). Sucrose solution (1.8 M sucrose, 3 mM MgAc2, 1 mM DTT, 10 mM Tris-HCl in DEPC-treated water) was added at the bottom of the tube and homogenates were centrifuged at 107,000 x g for 2.5 hours at 4°C. The supernatant was discarded, and the nuclei pellet was incubated in 200 μL of wash/resuspension buffer (WRB: 1% BSA, 0.2U/μL Protector RNAse inhibitor in DEPC-based PBS) for 20 min on ice before resuspending the pellet Gene expression and barcode libraries were prepared in parallel using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics) according to manufacturer's instructions. A detailed step-by-step protocol can be found on dx.doi.org/10.17504/protocols.io.kqdg369keg25/v2.